Numerous procedures employed in biomedical research and recombinant DNA technology and clinical diagnosis rely on nucleotides modified at the C-5 position for use as probes. These various utilities are based upon the ability of the molecules to be: (1) detected spectrophotometrically or (2) to form stable complexes with polypeptides which in turn can be detected, either by means of properties inherent in the polypeptide, or by means of detectable moieties which are attached to, or which interact with, the polypeptide.
Some uses include detecting and identifying nucleic acid-containing etiological agents, e.g. bacteria and viruses; screening bacteria for antibiotic resistance; diagnosing genetic disorders; and identifying tumor cells.
There are several important criteria which must be satisfied in order for a modified nucleotide to be generally suitable for use as probes in biomedical research, for clinical diagnosis and for recombinant DNA technology. The modified compound must contain a substituent or probe that is unique and not normally found associated with nucleotides or polynucleotides. The probe must react specifically with chemical or biological reagents to provide a sensitive detection system. The analogs may be relatively efficient substrates for commonly studied nucleic acid enzymes, since numerous practical applications require that the analog be enzymatically metabolized, e.g. the analogs must function as substrates for nucleic acid polymerases. For this purpose, probe moieties should not be placed on ring positions that sterically, or otherwise, interfere with the normal Watson-Crick hydrogen bonding potential of the bases. Otherwise, the substituents will yield compounds that are inactive as polymerase substrates. Normally such considerations limit substitution positions to the 5 position of a pyrimidine or pyrrolo[2,3-d]pyrimidine. Additionally, the detection system should be capable of interacting with probe substituents incorporated into both single-stranded and double-stranded polynucleotides in order to be compatible with nucleic acid hybridization methodologies. To satisfy this criteria, it is preferable that the probe moiety be attached to the pyrimidine through a chemical linkage or "linker arm" so that it can readily interface with antibodies, other detector proteins or chemical reagents.
C-5 substituted nucleosides are of seminal importance as modified components for (1) linking reactive functional group (e.g. sulfhydryl, amino, or carboxyl), biotin, fluorescent molecules, enzymes, or haptens to hybridization probe based diagnostics; (2) attaching radical generating metal complexes, crosslinking reagents, DNA cleaving reagents, and/or intercalators to sequence-specific oligonucleotides that are designed to function as therapeutic agents; and (3) linking fluorescent markers to oligonucleotides in automated DNA sequencing.
It can be seen therefore there is a real and continuing need for a convenient methodology for attaching C-5 linker arm substituents to the pyrimidine ring to allow nucleoside analogs to be prepared which will attach a variety of linker arms at the C-5 position to allow such useful compounds to be made faster, simpler, and in a more versatile manner from the standpoint of the precise linkages that may be attached at the C-5 position. Accordingly, it is a primary object of this invention to fulfill this continuing need.
Another objective of the present invention is to allow the addition of C-5 nucleoside substituents to be introduced by three different distinct strategies: First, C-5 substitution can be accomplished at the level of simple nucleosides in order to construct simple monomeric building blocks suitable for use in automated DNA synthesizers; secondly, modified nucleoside triphosphates can be constructed that will participate in enzyme reactions (DNA and RNA polymerases) that yield nucleic acids with the modification incorporated; and thirdly, DNA or RNA can be used as a probe by directly modifying via mercuration followed by a second reaction to attach the C-5 linker arm to the nucleic acid.
The manner of accomplishing each of the above objectives as well as others will become apparent from the detailed description of the invention which follows hereinafter.